The long-term goal of the proposed research is the identification and characterization of modes of gene expression unique to mitochondria. These studies define the possible mechanisms of gene expression and provide clues to the origin of genetic information. A unique step in mitochondrial gene expression has been identified in the mitochondria of the myxomycot, Physarum polycephalum. The genetic information for the alpha subunit of the ATP synthase complex is produced by insertion of cytidine residues at specific sites in the mRNA, a process termed RNA editing. The specific goal of this proposal is to determine the mechanism, extend and role of the RNA editing in mitochondrial gene expression in Physarum. The mechanism of RNA editing will be studied through the development of an in vitro editing system. Mitochondrial fractions having enzymatic activities associated with RNA editing will be purified and characterized. Mitochondrial nucleic acids will be screened for sequences which could serve as a template for this process, and the sequence of the mRNA will be systematically altered in order to determine its role in editing specificity. To determine the extent of RNA editing in Physarum mitochondria, additional genes on the mitochondrial DNA will be identified and screened to determine whether RNA editing is required for their expression. Finally, the role of RNA editing in the control of mitochondrial gene expression will be explored. Editing efficiencies during the life cycle and at the onset of plasmodial senescence will be compared, to determine if there is a correlation between RNA editing and mitochondrial activity in Physarum.